The identification and isolation of fluorescent proteins in various organisms, including marine organisms, has provided a valuable tool to molecular biology. The isolated fluorescent proteins have been used in their natural state and have been modified to elicit excitation/emission shifts in order to maximize their utility in in vitro and in vivo imaging. Green Fluorescent Proteins (GFPs) are the most widely utilized of the fluorescent proteins.
GFPs are involved in bioluminescence in a variety of marine invertebrates, including jellyfish such as Aequorea Victoria (Morise, H., et al., Biochemistry 13:2656-2662 (1974); Prendergast, F. G., and Mann, K. G., Biochemistry 17:3448-3453 (1978); Ward, W. W., Photochem. Photobiol. Rev. 4:1-57 (1979) and the sea pansy Renilla reniformis (Ward, W. W., and Cormier, M. J., Photochem. Photobiol. 27:389-396 (1978); Ward, W. W., et al., Photochem. Photobiol. 31:611-615 (1980)). The GFP isolated from A. victoria has been cloned and the primary amino acid structure has been deduced. The chromophore of A. victoria GFP is a hexapeptide composed of amino acid residues 64-69 in which the amino acids at positions 65-67 (serine, tyrosine and glycine) form a heterocyclic ring (Prasher, D. C., et al., Gene 111:229-233 (1992); Cody, C. W., et al., Biochemistry 32:1212-1218 (1993)). Resolution of the crystal structure of GFP has shown that the chromophore is contained in a central α-helical region surrounded by an 11-stranded β-barrel (Ormo, M., et al., Science 273:1392-1395 (1996); Yang, F., et al., Nature Biotech. 14:1246-1251 (1996)). Upon purification, native GFP demonstrates an absorption maximum at 395 nm and an emission maximum at 509 nm (Morise, H., et al., Biochemistry 13:2656-2662 (1974); Ward, W. W., et al., Photochem. Photobiol. 31:611-615 (1980)) with exceptionally stable and virtually non-photobleaching fluorescence (Chalfie, M., et al., Science 263:802-805 (1994)).
GFP has been used as a fluorescent label in protein localization and conformation studies and has been used as a reporter gene in transfected prokaryotic and eukaryotic cells (Heim, R., et al., Proc. Natl. Acad. Sci. USA 91:1250-1254 (1994); Yokoe, H., and Meyer, T., Nature Biotech. 14:1252-1256 (1996); Chalfie, M., et al., Science 263:802-805 (1994); Wang, S., and Hazelrigg, T., Nature 369:400-403 (1994)). GFP has also been used in fluorescence resonance energy transfer studies of protein-protein interactions (Heim, R., and Tsien, R. Y., Curr. Biol. 6:178-182 (1996)). Since GFP is naturally fluorescent, exogenous substrates and cofactors are not necessary for induction of fluorescence. Furthermore, the GFP cDNA containing the complete coding region is less than 1 kb and is easily manipulated and inserted into a variety of vectors for use in creating stable transfectants (Chalfie, M., et al., Science 263:802-805 (1994)). However, despite the relative ease at expressing the GFPs, they offer limited use for in vivo imaging of whole animals (e.g., mice and humans) because of the low absorption/emission maxima (395 nm/509 nm).
Accordingly, although the availability of a wide variety of naturally occurring fluorescent proteins and spectral variants of the proteins has allowed for substantial advances, limitations to the use of fluorescent proteins remain. In particular, the use of fluorescent proteins in intact animals such as mice has been hindered by poor penetration of excitation light. For example, visibly fluorescent proteins have been cloned from jellyfish and corals and have revolutionized many areas of molecular and cell biology through in vivo expression, in vitro expression, protein labeling, and protein engineering; however, the use of such fluorescent proteins for imaging studies in intact animals (e.g., a mouse or human) is limited due to the excitation and emission maxima of the fluorescent proteins.
Specifically, the excitation and emission maxima of these fluorescent proteins generally do not exceeded 598 and 655 nm respectively (D. Shcherbo et al., Nat. Methods 4, 741 (2007); M. A. Shkrob et al., Biochem. J. 392, 649 (2005); L. Wang, W. C. Jackson, P. A. Steinbach, R. Y. Tsien, Proc. Natl. Acad. Sci. U.S.A. 101, 16745 (2004)). One exception are the phytochrome-based fluorescent proteins that have an excitation maximum of 644 nm and an emission maximum of 672 nm (A. J. Fischer, J. C. Lagarias, Proc. Natl. Acad. Sci. U.S.A. 101, 17334 (2004)). However, neither the traditional fluorescent protein cloned from marine animals or the phytochrom-based fluorescent proteins are well equipped for in vivo imaging in whole, living animals.
In vivo optical imaging of deep tissues in animals is most feasible between 650 and 900 nm because such wavelengths minimize the absorbance by hemoglobin, water, and lipids as well as light scattering (F. F. Jobsis, Science 198, 1264 (1977)); R. Weissleder and V. Ntziachristos, Nat. Med. 9, 123 (2003)). Accordingly, the emission maximum of 598 and the absorption maximum of 655 nm of traditional fluorescent proteins (e.g., fluorescent proteins cloned from jellyfish and corals) are ineffective at in vivo optical imaging of deep tissues in animals. Thus, genetically encoded, infrared fluorescent proteins (IFPs) are particularly valuable for whole-body imaging in cancer, stem cell biology, gene therapy, and other areas of biomedical research and treatment.
IFPs provide an orthogonal color to GFP in protein labeling. Unlike the previously described dimeric Bacterial Phytochrome Photoreceptors (“BphPs”), the monomeric IFPs described can be used effectively to label proteins. Furthermore, instructions for engineering such monomeric IFPs is described herein that are based on a structural analysis of the dimer interface, the identification of a naturally occurring monomeric BphP from a BphP sequence database, and the protein modification of the naturally occurring BphP into a monomeric IFP (mIFP). Using the methods described herein, novel blue-shifted mutant mIFPs (iBlueberry) were designed and made. Exemplary iBlueberry mIFPs were designed with single mutations that introduce new thioether bonds between the chromophore and the protein. This new bond creates a twist in the chromophore which decreases conjugation efficiency, resulting in ˜40 nm blueshift. iBlueberry and mIFPs provide two orthogonal colors in protein labeling in cultured cells and model organisms and will find important applications in molecular and cell biology since biological processes such as cell signaling are carried out by many proteins forming dynamic network of protein-protein interactions.